Conformational changes in the antibody constant domains upon hapten-binding
Conformational changes in the antibody constant domains upon hapten-binding
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DOI：
10.1016/j.molimm.2004.07.004
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发表时间：
2005-01-01
影响因子：
3.6
通讯作者：
Azuma, T
中科院分区：
文献类型：
--
作者：
Sagawa, T;Oda, M;Azuma, T
细菌蛋白A和G （SpA和SpG）是免疫球蛋白受体，可作为监测重链常数构象变化的探针。(CH)域。采用等温滴定量热法和表面等离子体共振法测定了抗（4-羟基-3-硝基苯基）乙酰基（NP）抗体（Ab）与SpA和SpG的相互作用，以探讨半抗原结合是否引起CH结构域构象变化。在半抗原存在或不存在的情况下，测量IgG2a或其酶片段与SpA的相互作用。虽然没有观察到Fab和F(ab')(2)片段结合释放SpA，但它们确实结合了固定的SpA。此外，IgG2a与固定SpA相互作用的关联常数（K,）比与游离SpA相互作用高约20倍。这可以用IgG2a和芯片上固定的SpA之间的多价相互作用产生的高亲和度来解释。有趣的是，半抗原结合削弱了F（ab’）2片段与SpA之间的相互作用。此外，在半抗原存在的情况下，大约一半的IgG2a不能与固定的SpA结合。这些结果是用假设两种SpA/IgG复合物形成的模型来解释的；一个通过F(ab')2臂上的位点，另一个通过Fc区域的位点。前一种类型由于半抗原结合而解离，F(ab')2片段也是如此，这表明Fab臂周围发生了构象变化，而后一种类型由于Fc区域的高亲切度而没有解离。然而，使用具有较低K-a值的突变体SpA与IgG2a相互作用，表明半抗原结合诱导IgG2a Fc区域的远程构象变化。使用SpG作为探针也获得了半抗原结合后构象变化的类似证据。(C) 2004 Elsevier Ltd.版权所有。
Bacterial proteins A and G (SpA and SpG) are immunoglobulin receptors that can be used as probes for monitoring change in the conformation of heavy chain constant.(CH) domains. Interaction of anti-(4-hydroxy-3-nitrophenyi)acetyl (NP) antibody (Ab) with SpA and SpG were measured by isothermal titration calorimetry and surface plasmon resonance in order to address the question of whether hapten-binding induces a conformational change in the CH domain. The interactions of IgG2a or its enzymatic fragments with SpA were measured in the presence or absence of the hapten. Although binding of Fab and F(ab')(2) fragments were not observed to free SpA, they did bind to immobilized SpA. In addition, the association constant (K,) for interaction of IgG2a with immobilized SpA was approximately 20-fold higher than that with free SpA. This was explained in terms of high avidity resulting from multivalent interaction between IgG2a and immobilized SpA on the chip. Interestingly, the hapten-binding weakened the interaction between the F(ab')2 fragment and SpA. Furthermore, approximately half of the IgG2a was incapable of binding to immobilized SpA in the presence of hapten. These results were explained using a model which assumed the formation of two kinds of SpA/IgG complexes; one through sites on F(ab')2 arms and the other through sites on the Fc region. The former type dissociated as a result of hapten-binding, as did the F(ab')2 fragment and suggested that a conformational change had occurred around the Fab arms, while the latter type did not dissociate because of the higher avidity of the Fc region. However, using a mutant SpA with a lower K-a value for the interaction with IgG2a, it was shown that hapten-binding induced long range conformational changes in the Fc region of IgG2a. Similar evidence of conformational change upon hapten-binding was also obtained using SpG as a probe. (C) 2004 Elsevier Ltd. All rights reserved.